Background The ApoE4 genotype is the strongest genetic risk factor for developing AD. However, the mechanisms that underlie this link between ApoE4 genotype and SAD are not well understood. Objective/Hypothesis The objectives are to understand the molecular underpinnings of the association between ApoE4-specific changes in miRNA profile and ApoE4-induced brain phospholipid dysregulation which leads to ApoE4 increased susceptibility to develop SAD. Our recent findings demonstrate that ApoE proteins are critical determinants of brain phospholipid homeostasis and that the ApoE4 isoform is dysfunctional in this process. We have found that the levels of PIP2 are reduced in human and mouse brains of ApoE4 carriers, and in primary neurons expressing ApoE4 alleles when compared to those levels in ApoE3 counterparts. These changes are secondary to increased expression of a PIP2 degrading enzyme, the phosphoinositol phosphatase synaptojanin 1 (synj1), in ApoE4 carriers. Genetic reduction of synj1 in ApoE4 mice restores PIP2 levels and more importantly, rescues AD-related cognitive deficits. These findings are the first to link synj1 and PIP2 homeostasis to the pathogenic effects of ApoE4 in sporadic AD. Further studies suggest that ApoE4 behaves like the ApoE null conditions, which fails to degrade synj1 mRNA efficiently unlike ApoE3 does. We have also found that the levels of miR195 and miR374 are significantly lowered in ApoE4 mouse (9-12 months of age) and human brains (MCI and early AD subjects) compared to those in non-ApoE4 counterparts. Over- expression of miR195 but not miR374 in ApoE4 treated neurons reduces synj1 protein expression. Moreover, we have found that disruption of ApoE binding to its receptors LRP1 or genetic knockdown of LRP1 abolishes ApoE-induced changes in miR195/synj1 pathways. Therefore, we hypothesize that ApoE3 binds to LRP1 to up-regulate miR195 expression which subsequently modulates synj1 mRNA degradation rate and synj1 expression levels in the brain. In ApoE4 neurons and brains, decreased levels of miR195 may contribute to the reduced degradation rate of synj1 mRNA, thereby increasing synj1 protein expression and reduce brain PIP2 levels. These changes may contribute to ApoE4-associated synaptic and cognitive dysfunction. Rationale/Experimental Design In this application, we will study the effects of specific miRNA changes on ApoE4-associated phospholipid dyshomeostasis, synaptic dysfunction as well as cognitive dysfunction (aim 1). We will determine if elevating miR195 (which are low in ApoE4 mouse brains when compared to ApoE3 counterparts) by intraventricular injection of viral-containing miRNAs, can correct phospholipid dyshomeostasis, and rescue synaptic and cognitive deficits in ApoE4 mice without and with AD transgenic background in vivo. We will also determine if inhibiting miR195 by antagomirs in ApoE3 mouse brains without AD transgenic background can result in phospholipid dysregulation and subsequent synaptic and cognitive deficits, similarly to what we observed in ApoE4 mice. We propose to deliver miRNA-containing viral packages or miRNA antagomirs to hippocampal regions of 3-month old mice, and to determine their cognitive function at 6-9 months of age, as well as spine morphology and brain PIP2 homeostasis at 9-month of age. In aim 2, we propose to determine the signaling pathways that link ApoE isoforms to LRP1 leading to changes in miR195 and synj1 which contribute to ApoE4-related deficits. We propose to determine effects of miR195 on synj1 transcript by luciferase assays, to identify binding motifs of synj1 3?-UTR regions for miR195, to determine the downstream signal pathways from ApoE receptors to changes in miRNA and synj1 expression, and to identify key drives and candidate genes important in regulating brain phospholipid homeostasis and cognitive function after miRNA manipulations in ApoE mouse brains using NGS data assembly and network prediction analysis. Relevance/Impact The studies proposed in this application could help us design therapeutic interventions for the treatment of AD targeting at brain lipid homeostasis and ApoE4 through microRNA regulation.